Voltage-gated ion channels have evolved to open and close in response to changes in the membrane potential and rapidly conduct ions selectively. The members of the family that longest eluded isolation were the voltage-gated proton channel, Hv1, and its relatively close relative, the voltage sensing phosphatase (VSP). Hv1 plays a central role in innate immunity and other physiological processes. The biological function of VSP is not known. This proposal focuses on 3 fundamental aspects to the function of these VSD proteins, which, despite their similarities, differ radically in their effectors: with Hv1 having is channel effector uniquely situated within its VSD, while VSP's effector is the only one so far to have its effector outside of the membrane, in this case on the internal side. Our aims for Hv1 are to elucidate its pore pathway, understand how it is gated, how the gating apparatus in one subunit influences that of the dimeric partner and elucidate the mechanism by which Hv1 detects the absolute transmembrane gradient of pH and uses it to regulate gating. Our aim for VSP is to understand how conformational sequences in the VSD induce conformational sequences in the enzyme domain to alter the choice of substrate. Our goal is to arrive at mechanistic molecular models of gating, cooperativity and modulation of the VSD by pH and modulation by the VSD of the effector. The proposed studies should provide insight into the function of VSDs across voltage-gated proteins and the new methods should be applicable to a range of other channels and receptors whose protein motions, subunit interactions and modulation by ligands are of interest. The proposed work is designed to elucidate the mechanism of function of the voltage-gated proton channel (which is fundamental to innate immunity, reproduction and epithelial transport, and appears to have a role in stroke and cancer) and its relative, the voltage sensing phosphatase. The approach employs several novel optical methods that should prove to be applicable to the study of a broad set of ion channels and receptors.